Adenosine-3′:5′-cyclic monophosphate (3′:5′-cAMP) is the second messenger mediating the transmembrane actions of numerous agents throughout the animal kingdom. It acts typically through the activation of 3′:5′-cAMP-dependent protein kinases (PKA), which catalyze the phosphorylation of specific proteins [1,2] or through effects on 3′:5′-cAMP-gated ion channels [3,4]. Consequently, relationships between 3′:5′-cAMP and cell function are typically viewed as effects mediated by these two mechanisms. The 3′:5′-cAMP signaling pathway can be regulated pharmacologically by many drugs that are of particular value in the treatment of various diseases and therefore there is continuing interest in identifying new agents acting on this pathway.
Cellular levels of 3′:5′-cAMP are regulated by adenylyl cyclase, a family of membrane-bound enzymes that catalyze the formation of 3′:5′-cAMP from 5′-ATP [5,6], and cyclic nucleotide phosphodiesterases, a family of enzymes that catalyze conversion of 3′:5′-cAMP to 5′-AMP [7,8]. Agents that affect cellular 3′:5′-cAMP levels, whether by affecting the activity of cyclic nucleotide phosphodiesterases or of adenylyl cyclases, also will typically affect changes in cell function.
Mammalian forms of adenylyl cyclase are a family of membrane-bound enzymes that catalyze the formation of 3′:5′-cAMP from 5′-ATP. The family includes at least 10 isozymes and their expression is tissue dependent and developmentally variable. Activities of adenylyl cyclases are regulated by numerous neurotransmitters, autacoids, and hormones via cell surface receptors that act through heterotrimeric guanine nucleotide-dependent regulatory proteins (G-proteins; comprising α, β, and γ subunits). G-proteins may be either stimulatory (G,) [9] or inhibitory (Gi) for adenylyl cyclase activity, and may be either specific or promiscuous in effecting activity of different isozymes [9-11]: GSα, for example, activates all isozymes, possibly save the isozyme from sperm. Adenylyl cyclase activity may also be altered by other agents of physiological and biochemical interest, including bacterial toxins that act on GS and Gi, agents or enzymes that act on hormone receptors, and agents that act directly on adenylyl cyclase, e.g. Ca2+/calmodulin [12], forskolin [13,14], certain oxidants [15,16], protein kinases [17], and various adenine derivatives.
Numerous drugs have been developed as therapeutic agents that inhibit cyclic nucleotide phosphodiesterases [8]. There are also numerous therapeutically useful drugs that activate or inhibit adenylyl cyclases indirectly. One class of therapeutic agents having an indirect effect on adenylyl cyclases includes agents that are receptor blockers, such as agents that, for example, block receptors for catecholamines (dopamine, norepinephrine, and epinephrine), angiotensin II, adenosine and other purines. B-blockers that are commonly used to treat hypertension, for example, act to inhibit adenylyl cyclase indirectly by blocking the stimulatory effects of the sympathetic nervous system to activate adenylyl cyclase in the heart [5], in which the type V isozyme is expressed predominantly [6]. By comparison to drugs that inhibit cyclic nucleotide phosphodiesterases or that affect adenylyl cyclases indirectly, drugs that act directly on adenylyl cyclases are uncommon. The main class of such agents comprises analogs of forskolin [13,14].
Drugs that inhibit adenylyl cyclase directly would be particularly highly prized. The expectation is that such agents that act downstream of cell-surface receptors to inhibit adenylyl cyclase directly should have a cardiac-sparing effect and be useful in reducing cardiomyopathies and heart failure. We have developed a means to block the formation of 3′:5′-cAMP in intact tissues by a unique class of specific and selective nucleotide prodrugs. These nucleotide prodrugs will find extensive use as pharmacologically and biochemically useful inhibitors of adenylyl cyclase. The prodrugs exhibit potencies in the nanomolar range and have shown their usefulness to regulate function in isolated cells and in intact tissues. Thus, the prodrugs of the invention are the most potent, directly acting inhibitors of adenylyl cyclases in tissues, and represent a new and unique class of drug with therapeutic potential.
Adenylyl cyclases are inhibited by a number of polyphosphorylated derivatives of adenine and adenosine (Table 1). Biochemical assays have been used to characterize inhibition of adenylyl cyclases by adenine derivatives with respect to ligand structures and inhibition kinetics [18-35]. The enzyme is inhibited competitively by substrate analogs that interact with a pre-transition configuration of the enzyme [31]. The most potent of these is β-L-2′,3′-dd-5′-ATP (Table 1). In contrast, inhibition by adenine nucleoside 3′-phosphates bind to a post-transition configuration and inhibition is uncompetitive or non-competitive, depending on the mode of activation [25,26,33]. Potent inhibitors that bind selectively to pre- and post-transition states are extremely uncommon and because of this these compounds may form the basis of new inhibitors with unique biochemical and pharmacological properties.
TABLE 1IC50s for rat brain adenylyl cyclaseNUCLEOSIDE (IC50, μM)3′-PhosphateAdo2′dAdo2′,5′ddAdonone82152.73′˜P8.91.20.463′˜PP3.90.140.103′˜PPP2.00.090.0403′˜PPPP—0.01050.00743′˜PS—3.10.60Substrate analogs:IC50 (μM)β-L-5′-AMP200β-L-2′,3′-dd-5′-AMP62β-L-5′-ATP3.2β-L-2′,3′-dd-5′-ATP0.024PMEA-mimics:IC50 (μM)PMEA65PMEApp0.17PMEAp(NH)p0.18From Johnson and coll. [27-32]
The acyclic phosphonate derivatives of adenine, typified by PMEApp [32], are somewhat less potent than β-L-2′,3′-dd-5′-ATP or 2′,5′-dd-3′-ATP (Table 1), but the kinetic behavior of these agents conformed to mixed inhibition, i.e., kinetic behavior sharing aspects with that seen with the competitive and noncompetitive inhibitors. These distinctions form the basis for the rational design of compounds that can effect inhibition of adenylyl cyclases in intact tissues.
Crystal structures of the adenylyl cyclase catalytic cleft were solved in both pre- and post-transition states by use of β-L-2′,3′-dd-5′-ATP and 2′,5′dd-3′-ATP [36,37]. Note that there are only small differences in the topology of the catalytic cleft into which the respective ligands bind. It is presumed that PMEApp and its mimics bind in this same locus.
There are a number of cell-permeable derivatives of adenine that have been used to lower cellular 3′:5′-cAMP levels and to alter function in isolated cells and intact tissues. These include 2′,5′-dideoxyadenosine (2′,5′-dd-Ado), 9-(tetrahydrofuryl)-adenine (9-THF-Ade;), 9-(arabinofurnosyl)-adenine (9-Ana-Ade), or 9-(cyclopentyl)-adenine (9-CP-Ate). These compounds am typically effective at concentrations from 10 to 100 μM. They have been used with epididymal fat cells [38], isolated hepatocytes [39], pre-adipocytes [40], thyroid follicles [41], dorsal root ganglion neurons [42], bone organ cultures [43], cortical collecting tubules [44], phagocytes [45], and platelets [20], to name but a few. End-points included cell differentiation [40], water conductance [44], action potential after-hyperpolarization [42], bone resorption [43], glycerol production [38], altered enzyme activity [39], DNA synthesis and cell growth [41], and FCγ-receptor-mediated phagocytosis [45]. Effects of these agents on cell function were uniformly consistent with inhibition of adenylyl cyclase by these ligands. The expectation is that the compounds of the invention will also affect cell function in these systems, but be substantially more potent in doing so.